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Purification of a Drug Substance of a Factor VII Polypeptide by Removal of DesGla-Factor VII Polypeptide Structures

a polypeptide and purification process technology, applied in the direction of peptides, peptide/protein ingredients, immunoglobulins, etc., can solve the problems of inability to meet the needs of patients,

Inactive Publication Date: 2009-02-12
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0001]The present invention relates to a purification process for drug substances of a Factor VII polypeptide having an impurity in the form of desGla-Factor VII polypeptide structures. The process utilizes an anion-exchange material and includes washing and / or elution with a buffer of a predetermined pH.

Problems solved by technology

The overall industrial-scale process for the purification of drug substances of a Factor VII polypeptides may in certain instances suffer from the drawback that the drug substance comprises a considerable amount of corresponding desGla-Factor VII polypeptide structures, i.e. the drug substance is considered to include a considerable amount of an impurity.
This is—of course—undesirable and in some instances also unacceptable.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Determination of Content of desGla-Factor VII Polypeptide Structures

[0109]The content of desGla-Factor VII polypeptide structures relative to the full length Factor VII polypeptide structures is determined by SDS-PAGE. 150 μl of sample is added 50 μl of sample buffer (non reducing, NuPAGE) and boiled for 5 mins. A 10 μl sample is loaded onto a 12% BisTris NuPAGE Gel (Invitrogen). The gel is run at 200 Volts, 120 mA for 55 mins. The gel is stained using coomassie brilliant blue solution, destained and dried. The relative desGla-Factor VII polypeptide content is calculated as the area of the desGla-Factor VII polypeptide band divided by the areas of the Factor VII polypeptide band at approx. 50 kDa and desGla-Factor VII polypeptide band at approx. 45 kDa.

example a

[0110]Anion exchange chromatography is performed on a column (1 cm inner diameter×10 cm length=7.85 ml column volume(CV)) packed with Pharmacia Q-Sepharose Fast Flow, equilibrated with 5 CV of a solution containing 5 mM EDTA, 10 mM histidine, pH 6.0. The load is 40 CVs of a filtered solution containing 1 mg / ml FVIIa analogue including 12% desGla-Factor VII polypeptide structures, followed by a 5 CV wash using 50 mM NaCl, 10 mM histidine, pH 6.0. The elution is performed using a 10 CV gradient from 25 mM NaCl to 50 mM CaCl2, 25 mM NaCl, buffered at pH 6.0 by 10 mM histidine. The entire purification is carried out at a flow-rate of 20 CV / h and a temperature of 5° C.

[0111]The product peak elutes approximately at 12 mM CaCl2. The eluate is analysed by SDS-PAGE which will indicate a content of desGla-Factor VII polypeptide structure of less than 2% (below the limit of detection).

example b

[0112]Anion exchange chromatography is performed on a column (1 cm inner diameter×10 cm length=7.85 ml column volume(CV)) packed with Pharmacia Q-Sepharose Fast Flow, equilibrated with 5 CV of a solution containing 5 mM tri sodium citrate, 10 mM tris, pH 8.0. The load is 40 CVs of a filtered solution containing 1 mg / ml FVIIa analogue including 12% desGla-Factor VII polypeptide structures, followed by a 5 CV wash using 50 mM NaCl, 10 mM tris, and pH 8.0. The elution is performed using a 20 CV gradient from 50 mM NaCl to 750 mM NaCl, buffered at pH 6.0 by 10 mM histidine. The entire purification is carried out at a flow-rate of 10 CV / h and a temperature of 5° C.

[0113]The product peak elutes approximately at 350 mM NaCl. The eluate is analysed by SDS-PAGE which will indicate a content of desGla-Factor VII polypeptide structure of less than 2% (below the limit of detection).

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Abstract

The present invention relates to a purification process for drug substances of a Factor VII polypeptide having an impurity in the form of desGla-Factor VII polypeptide structures. The process utilizes an anion-exchange material and includes washing and / or elution with a buffer of a predetermined pH.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a purification process for drug substances of a Factor VII polypeptide having an impurity in the form of desGla-Factor VII polypeptide structures. The process utilizes an anion-exchange material and includes washing and / or elution with a buffer of a predetermined pH.BACKGROUND OF THE INVENTION[0002]The proteins involved in the clotting cascade, including, e.g., Factor VII, Factor VIII, Factor IX, Factor X, and Protein C, are proving to be useful therapeutic agents to treat a variety of pathological conditions. Accordingly, there is an increasing need for formulations comprising these proteins that are pharmaceutically acceptable and exhibit a uniform and predetermined clinical efficacy.[0003]The overall industrial-scale process for the purification of drug substances of a Factor VII polypeptides may in certain instances suffer from the drawback that the drug substance comprises a considerable amount of corresponding desGla...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/37
CPCC12Y304/21021C12N9/6437
Inventor KRARUP, JANUS
Owner NOVO NORDISK AS
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